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Rock Hill, United States

Yo J.,MultiGEN Diagnostics LLC | Hay K.S.L.,MultiGEN Diagnostics LLC | Vinayagamoorthy D.,MultiGEN Diagnostics LLC | Maryanski D.,MultiGEN Diagnostics LLC | And 3 more authors.
MethodsX | Year: 2015

Abstract Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal.Tumorplex™ provides a high level of sensitivity.Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA. © 2015 The Authors. Source

Vinayagamoorthy T.,MultiGEN Diagnostics LLC | Maryanski D.,MultiGEN Diagnostics LLC | Vinayagamoorthy D.,MultiGEN Diagnostics LLC | Hay K.S.L.,MultiGEN Diagnostics LLC | And 3 more authors.
MethodsX | Year: 2015

Abstract The two principal determining steps in molecular diagnosis are the amplification and the identification steps. Accuracy of DNA amplification is primarily determined by the annealing sequence of the PCR primer to the analyte DNA. Accuracy for identification is determined either by the annealing region of a labelled probe for the real time PCR analysis, or the annealing of a sequencing primer for DNA sequencing analysis, that binds to the respective analyte (amplicon). Presently, housekeeping genes (Beta globin, GAPDH) are used in molecular diagnosis to verify that the PCR conditions are optimum, and are thus known as amplification controls [1-4]. Although these genes have been useful as amplification controls, they lack the true definition of an internal control because the primers and annealing conditions are not identical to the analyte being assayed. This may result in a false negative report [5]. The IC-Code platform technology described here provides a true internal control where the internal control and analyte share identical PCR primers annealing sequences for the amplification step and identical sequencing primer annealing sequence for the identification step. The analyte and internal control have the same PCR and sequencing annealing sequences.This method assures for little or no false negatives and false positives due to the method's design of using identical annealing conditions for the internal control and analyte, and by using DNA sequencing analysis for the identification step of the analyte, respectively.This method also allows for a set lower limit of detection to be used by varying the amount of internal control used in the assay. © 2015 The Authors. Published by Elsevier B.V. Source

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